The goal of our research program is to understand the mechanisms which underlie the differences in the regulation of cell proliferation between the normal human melanocyte and the metastatic melanoma cell. Malignant melanoma is increasing very rapidly in the united States. Prognosis is good prior to metastasis but for metastatic melanoma the outlook is grim. The molecular basis of the malignancy is not well understood. Phorbol esters stimulate the growth of normal melanocytes but they inhibit the growth of most metastatic melanoma cells. The consistency of this difference in signal transduction pathways suggests that it may be a major determinant of the malignant phenotype. Our primary focus is to understand how the molecular mechanisms by which phorbol esters regulate cellular proliferation are disturbed in metastatic melanoma cells. Our initial observations demonstrated that addition of 12-O-tetradecanoyl phorbol-13- acetate (TPA) to metastatic melanoma cells leads to growth arrest in both G1 and G2 phases of the cell cycle. To understand the mechanism regulating the sequence of events leading to this biphasic growth arrest experiments are planned a) to map the TPA restriction points in the cell cycle using flow microfluorimetry in conjunction with specific inhibitors of protein kinase C; b) to analyze how TPA alters the phosphorylation, synthesis and enzymatic activity of the cell cycle control proteins p34 cdc2, CDK2 and associated cyclins; and c) to analyze how TPA alters the levels of p34 cdc2, CDK2 and cyclin mRNAs. The proposed experiments ar designed to increase our understanding of the mechanisms regulating proliferation which are altered when the normal melanocyte is transformed into a metastatic melanoma cell. Because the growth of many malignant cell lies is inhibited by phorbol esters, this analysis of growth arrest in melanoma cells may have broad implications for the rational development of drugs designed specifically to halt the growth of several tumor cell types.